Chapter 6Chapter 6

Microbial GrowthMicrobial Growth

Bacterial Cell DivisionBacterial Cell Division

New cells are formed by cell New cells are formed by cell fissionfission

Cells do not grow – they double Cells do not grow – they double their cytoplasmic contents and their cytoplasmic contents and membranemembrane

They synthesize essential They synthesize essential molecules needed for their molecules needed for their metabolic processesmetabolic processes

Binary Fission in Binary Fission in BacteriaBacteria

PartitioningPartitioning

Prior to cell division, Prior to cell division, bacteria copy their bacteria copy their DNA( replicate their DNA)DNA( replicate their DNA)

They then partition the They then partition the DNA by constructing a cell DNA by constructing a cell wall between the two wall between the two molecules of DNAmolecules of DNA

This insures that the new This insures that the new cell receives a copy of the cell receives a copy of the chromosomechromosome

The division or partitioning The division or partitioning of chromosomes is more of chromosomes is more difficult in those organisms difficult in those organisms that have more than one that have more than one chromosomechromosome

Prokaryote vs. Prokaryote vs. EukaryoteEukaryote Prokaryote cells do not go Prokaryote cells do not go

through the cell cycle like through the cell cycle like eukaryote cellseukaryote cells

They divide by fissionThey divide by fission In some species there is some In some species there is some

linkage which forms tetrads, linkage which forms tetrads, sarcinae, and even staphylococcisarcinae, and even staphylococci

GrowthGrowth Increase in cellular constituents that Increase in cellular constituents that

may result in:may result in:– increase in cell numberincrease in cell number

when microorganisms reproduce by budding or when microorganisms reproduce by budding or binary fissionbinary fission

– increase in cell sizeincrease in cell size coenocyticcoenocytic microorganisms have nuclear divisions microorganisms have nuclear divisions

that are not accompanied by cell divisions. Fungi that are not accompanied by cell divisions. Fungi have a syncytium and their nuclei are not have a syncytium and their nuclei are not separated.separated.

Microbiologists usually study population Microbiologists usually study population growth rather than growth of individual growth rather than growth of individual cellscells

The Growth CurveThe Growth Curve

Observed when microorganisms Observed when microorganisms are cultivated in are cultivated in batch culturebatch culture– culture incubated in a closed culture incubated in a closed

vessel with a single batch of vessel with a single batch of mediummedium

Usually plotted as logarithm of Usually plotted as logarithm of cell number versus timecell number versus time

Usually has four distinct phasesUsually has four distinct phases

Figure 6.1

no increase

maximal rate of divisionand population growth

population growth ceases

decline inpopulationsize

Lag PhaseLag Phase

Cell synthesizing new Cell synthesizing new componentscomponents– to replenish spent materialsto replenish spent materials– to adapt to new medium or other to adapt to new medium or other

conditionsconditions varies in lengthvaries in length

– in some cases can be very short or in some cases can be very short or even absenteven absent

Exponential PhaseExponential Phase

Also called Also called log phaselog phase Rate of growth is constantRate of growth is constant Population is most uniform in Population is most uniform in

terms of chemical and physical terms of chemical and physical properties during this phaseproperties during this phase

cells are dividing and doubling in number at regular intervals

Each individualcell divides at aslightly differenttime

Curve risessmoothly ratherthan as discretesteps

E. ColiE. Coli Growth Curve Growth Curve

Balanced growthBalanced growth

During log phase, cells exhibit During log phase, cells exhibit balanced growthbalanced growth– cellular constituents manufactured cellular constituents manufactured

at constant rates relative to each at constant rates relative to each otherother

Unbalanced growthUnbalanced growth

Rates of synthesis of cell Rates of synthesis of cell components vary relative to each components vary relative to each otherother

Occurs under a variety of conditionsOccurs under a variety of conditions– change in nutrient levelschange in nutrient levels

shift-up (poor medium to rich medium)shift-up (poor medium to rich medium) shift-down (rich medium to poor medium)shift-down (rich medium to poor medium)

– change in environmental conditionschange in environmental conditions

Stationary PhaseStationary Phase

total number of viable cells total number of viable cells remains constantremains constant– may occur because metabolically may occur because metabolically

active cells stop reproducingactive cells stop reproducing– may occur because reproductive may occur because reproductive

rate is balanced by death raterate is balanced by death rate

Possible reasons for Possible reasons for entry into stationary entry into stationary phasephase

nutrient limitationnutrient limitation limited oxygen availabilitylimited oxygen availability toxic waste accumulationtoxic waste accumulation critical population density critical population density

reachedreached

Starvation responsesStarvation responses

morphological changesmorphological changes– endospore formationendospore formation

decrease in size, protoplast decrease in size, protoplast shrinkage, and nucleoid shrinkage, and nucleoid condensationcondensation

production of starvation proteinsproduction of starvation proteins long-term survivallong-term survival increased virulenceincreased virulence

Death PhaseDeath Phase

cells dying, usually at exponential cells dying, usually at exponential raterate

deathdeath– irreversible loss of ability to irreversible loss of ability to

reproducereproduce in some cases, death rate slows in some cases, death rate slows

due to accumulation of resistant due to accumulation of resistant cellscells

The Mathematics of The Mathematics of GrowthGrowth Generation (doubling) timeGeneration (doubling) time

– time required for the population to time required for the population to double in sizedouble in size

Mean growth rate constantMean growth rate constant– number of generations per unit timenumber of generations per unit time– usually expressed as generations usually expressed as generations

per hourper hour

The Generation TimeThe Generation Time

The generation time for most species The generation time for most species is between twenty minutes and 24 is between twenty minutes and 24 hours.hours.

Some organisms take a longer time to Some organisms take a longer time to go through the lag phasego through the lag phase

Some organisms due to their Some organisms due to their characteristics like characteristics like Mycobacterium Mycobacterium tuberculosistuberculosis grow slowly due to the grow slowly due to the cell wallcell wall

Synchronous GrowthSynchronous Growth

Cells doubling or dividing every Cells doubling or dividing every 20 minutes20 minutes

Measurement of Measurement of Microbial GrowthMicrobial Growth

Can measure changes in number Can measure changes in number of cells in a populationof cells in a population

Can measure changes in mass of Can measure changes in mass of populationpopulation

Measurement of Cell Measurement of Cell NumbersNumbers

Direct cell countsDirect cell counts– counting chamberscounting chambers– electronic counterselectronic counters– on membrane filterson membrane filters

Viable cell countsViable cell counts– plating methodsplating methods– membrane filtration methodsmembrane filtration methods

Counting chambersCounting chambers

easy, easy, inexpensive, inexpensive, and quickand quick

useful for useful for counting both counting both eucaryotes and eucaryotes and procaryotesprocaryotes

cannot cannot distinguish distinguish living from dead living from dead cellscells Figure 6.5

Electronic countersElectronic counters

microbial suspension forced microbial suspension forced through small orificethrough small orifice

movement of microbe through movement of microbe through orifice impacts electric current orifice impacts electric current that flows through orificethat flows through orifice

instances of disruption of current instances of disruption of current are countedare counted

Electronic counters…Electronic counters…

cannot distinguish living from cannot distinguish living from dead cellsdead cells

quick and easy to usequick and easy to use useful for large microorganisms useful for large microorganisms

and blood cells, but not and blood cells, but not procaryotesprocaryotes

Direct counts on Direct counts on membrane filtersmembrane filters cells filtered through special cells filtered through special

membrane that provides dark membrane that provides dark background for observing cellsbackground for observing cells

cells are stained with fluorescent cells are stained with fluorescent dyesdyes

useful for counting bacteriauseful for counting bacteria with certain dyes, can distinguish with certain dyes, can distinguish

living from dead cellsliving from dead cells

Plating methodsPlating methods

Measure Measure number of number of viable cellsviable cells

Population Population size is size is expressed expressed as as colony colony forming forming unitsunits (CFU)(CFU)

plate dilutions of plate dilutions of population on population on suitable solid suitable solid

mediummedium

count number of count number of

coloniescolonies

calculate number of calculate number of

cells in populationcells in population

Spread PlateSpread Plate

Samples are diluted by using 1 ml of Samples are diluted by using 1 ml of broth culture and 9 ml of sterile nutrient broth culture and 9 ml of sterile nutrient brothbroth

MixMix Then 1 ml of the 1:10 ( first dilution) is Then 1 ml of the 1:10 ( first dilution) is

added to another 9ml of fresh nutrient added to another 9ml of fresh nutrient brothbroth

MixMix Samples are diluted by using 1ml of broth Samples are diluted by using 1ml of broth

culture and 9 ml of sterile nutrient brothculture and 9 ml of sterile nutrient broth MixMix

Standard DilutionsStandard Dilutions

Spread plateSpread plate

A ml of each dilution is pipetted A ml of each dilution is pipetted with a plastic transfer pipet to the with a plastic transfer pipet to the center of an agar platecenter of an agar plate

A spreader( looks like a hockey A spreader( looks like a hockey stick) is used to spread the cells stick) is used to spread the cells across the surfaceacross the surface

This is designed to produce an This is designed to produce an even distribution throughouteven distribution throughout

Colony CounterColony Counter

Colony CounterColony Counter

To make an exact count of the colonies you To make an exact count of the colonies you place the plate on a gridplace the plate on a grid

You then illuminate the plate.You then illuminate the plate. You count the colonies in the grid by going You count the colonies in the grid by going

across a horizontal row and then vertically to across a horizontal row and then vertically to the next row until you have covered the whole the next row until you have covered the whole plateplate

The final count is multiplied x the dilution The final count is multiplied x the dilution factor. This number is the number of bacteria factor. This number is the number of bacteria that were in 1 ml of culturethat were in 1 ml of culture

It is assumed that each colony is equal to 1 It is assumed that each colony is equal to 1 original cell in the broth cultureoriginal cell in the broth culture

Applications of this Applications of this technique commonly technique commonly used in the laboratoryused in the laboratory Determination of coliforms in the Determination of coliforms in the

environment( E. coli)environment( E. coli) Determination of cells Determination of cells

transformed by genetic transformed by genetic engineeringengineering

Determination of bacteria Determination of bacteria contaminating soil in the contaminating soil in the environmentenvironment

Problems with colony Problems with colony counts using platescounts using plates There is error in this methodThere is error in this method If the dilutions are homogeneous, If the dilutions are homogeneous,

there can be errorsthere can be errors This may not capture all This may not capture all

organisms in a broth because organisms in a broth because some may not be able to grow on some may not be able to grow on the chosen mediathe chosen media

Colony CountsColony Counts

Pour PlatesPour Plates

Add 1 ml of a serial Add 1 ml of a serial dilution to 9 ml of dilution to 9 ml of melted and slightly melted and slightly warm nutrient agarwarm nutrient agar

MixMix Pour into a Petri dish Pour into a Petri dish

and allow it to hardenand allow it to harden Colonies will develop Colonies will develop

both in the media and both in the media and on the mediaon the media

Cells may be damaged Cells may be damaged by the hot agar in this by the hot agar in this experimentexperiment

Plating methods…Plating methods…

simple and sensitivesimple and sensitive widely used for viable counts of widely used for viable counts of

microorganisms in food, water, microorganisms in food, water, and soiland soil

inaccurate results obtained if cells inaccurate results obtained if cells clump togetherclump together

Most Probable NumberMost Probable Number

Most probable number is used for Most probable number is used for environmental samplesenvironmental samples

Trying to determine the presence of an Trying to determine the presence of an organismorganism

Use dilution factors as previously describedUse dilution factors as previously described Use multiple tubes for dilutionsUse multiple tubes for dilutions Check broth for cloudiness or turbidity( signs Check broth for cloudiness or turbidity( signs

of bacterial growth)of bacterial growth) Use culture tubes containing sugars( lactose, Use culture tubes containing sugars( lactose,

sucrose, glucose) These can be checked for sucrose, glucose) These can be checked for the presence of gas with a small tube on the the presence of gas with a small tube on the interior called a Durham tube.interior called a Durham tube.

See chart on page 149 for clarificationSee chart on page 149 for clarification

Membrane filtration Membrane filtration methodsmethods

Figure 6.6especially useful for analyzing aquatic samples

Measurement of Cell Measurement of Cell MassMass

dry weightdry weight– time consuming and not very sensitivetime consuming and not very sensitive

quantity of a particular cell constituentquantity of a particular cell constituent– protein, DNA, ATP, or chlorophyllprotein, DNA, ATP, or chlorophyll– useful if amount of substance in each cell is useful if amount of substance in each cell is

constantconstant turbidometric measures (light turbidometric measures (light

scattering)scattering)– quick, easy, and sensitivequick, easy, and sensitive

Figure 6.8

more cells

more lightscattered

less lightdetected

The Continuous Culture The Continuous Culture of Microorganismsof Microorganisms

growth in an open systemgrowth in an open system– continual provision of nutrientscontinual provision of nutrients– continual removal of wastescontinual removal of wastes

maintains cells in log phase at a maintains cells in log phase at a constant biomass concentration constant biomass concentration for extended periodsfor extended periods

achieved using a achieved using a continuous continuous culture systemculture system

The ChemostatThe Chemostat

rate of incoming rate of incoming medium = rate medium = rate of removal of of removal of medium from medium from vesselvessel

an essential an essential nutrient is in nutrient is in limiting limiting quantitiesquantities

Figure 6.9

Dilution rate and Dilution rate and microbial growthmicrobial growth

Figure 6.10

dilution rate – rate atwhich medium flowsthrough vesselrelative to vessel size

note: cell densitymaintained at widerange of dilutionrates and chemostat operates best at low dilution rate

The TurbidostatThe Turbidostat

regulates the flow rate of media regulates the flow rate of media through vessel to maintain a through vessel to maintain a predetermined turbidity or cell predetermined turbidity or cell densitydensity

dilution rate variesdilution rate varies no limiting nutrientno limiting nutrient turbidostat operates best at high turbidostat operates best at high

dilution ratesdilution rates

Importance of Importance of continuous culture continuous culture methodsmethods

constant supply of cells in exponential constant supply of cells in exponential phase growing at a known ratephase growing at a known rate

study of microbial growth at very low study of microbial growth at very low nutrient concentrations, close to nutrient concentrations, close to those present in natural environmentthose present in natural environment

study of interactions of microbes study of interactions of microbes under conditions resembling those in under conditions resembling those in aquatic environmentsaquatic environments

food and industrial microbiologyfood and industrial microbiology

The Influence of The Influence of Environmental Factors Environmental Factors on Growthon Growth

most organisms grow in fairly most organisms grow in fairly moderate environmental moderate environmental conditionsconditions

extremophilesextremophiles– grow under harsh conditions that grow under harsh conditions that

would kill most other organismswould kill most other organisms

Solutes and Water Solutes and Water ActivityActivity water activity (awater activity (aww))

– amount of water available to amount of water available to organismsorganisms

– reduced by interaction with solute reduced by interaction with solute molecules (osmotic effect)molecules (osmotic effect)

higher [solute] higher [solute] lower a lower aww

– reduced by adsorption to surfaces reduced by adsorption to surfaces (matric effect)(matric effect)

Osmotolerant Osmotolerant organismsorganisms grow over wide ranges of water activitygrow over wide ranges of water activity

many use many use compatible solutescompatible solutes to to increase their internal osmotic increase their internal osmotic concentrationconcentration– solutes that are compatible with metabolism solutes that are compatible with metabolism

and growthand growth some have proteins and membranes some have proteins and membranes

that require high solute concentrations that require high solute concentrations for stability and activityfor stability and activity

Effects of NaCl on Effects of NaCl on microbial growthmicrobial growth halophileshalophiles

– grow optimally at grow optimally at >0.2 M>0.2 M

extreme extreme halophileshalophiles– require >2 Mrequire >2 M

Figure 6.11

pHpH

negative negative logarithm of the logarithm of the hydrogen ion hydrogen ion concentrationconcentration

pHpH

acidophilesacidophiles– growth optimum between pH 0 and pH 5.5growth optimum between pH 0 and pH 5.5

neutrophilesneutrophiles– growth optimum between pH 5.5 and pH 7growth optimum between pH 5.5 and pH 7

alkalophilesalkalophiles– growth optimum between pH8.5 and pH growth optimum between pH8.5 and pH

11.511.5

pHpH most acidophiles and alkalophiles most acidophiles and alkalophiles

maintain an internal pH near neutralitymaintain an internal pH near neutrality– some use proton/ion exchange mechanisms to some use proton/ion exchange mechanisms to

do sodo so some synthesize proteins that provide some synthesize proteins that provide

protectionprotection– e.g., acid-shock proteinse.g., acid-shock proteins

many microorganisms change pH of their many microorganisms change pH of their habitat by producing acidic or basic waste habitat by producing acidic or basic waste productsproducts– most media contain buffers to prevent growth most media contain buffers to prevent growth

inhibitioninhibition

TemperatureTemperature

organisms organisms exhibit exhibit distinct distinct cardinal cardinal growth growth temperaturestemperatures– minimalminimal– maximalmaximal– optimaloptimal Figure 6.13

Figure 6.14

Temperature and Temperature and bacterial growthbacterial growth

Adaptations of Adaptations of thermophilesthermophiles

protein structure stabilized by a variety protein structure stabilized by a variety of means of means – more H bondsmore H bonds– more prolinemore proline– chaperoneschaperones

histone-like proteins stabilize DNAhistone-like proteins stabilize DNA membrane stabilized by variety of membrane stabilized by variety of

meansmeans– more saturated, more branched and higher more saturated, more branched and higher

molecular weight lipidsmolecular weight lipids– ether linkages (archaeal membranes)ether linkages (archaeal membranes)

Oxygen Oxygen ConcentrationConcentration

Figure 6.15

needoxygen

preferoxygen

ignoreoxygen

oxygen istoxic

< 2 – 10%oxygen

Basis of different Basis of different oxygen sensitivitiesoxygen sensitivities oxygen easily reduced to toxic oxygen easily reduced to toxic

productsproducts– superoxide radical superoxide radical – hydrogen peroxidehydrogen peroxide– hydroxyl radicalhydroxyl radical

aerobes produce protective enzymesaerobes produce protective enzymes– superoxide dismutase (SOD)superoxide dismutase (SOD)– catalasecatalase

Figure 6.14

PressurePressure

barotolerant organismsbarotolerant organisms– adversely affected by increased adversely affected by increased

pressure, but not as severely as pressure, but not as severely as nontolerant organismsnontolerant organisms

barophilic organismsbarophilic organisms– require or grow more rapidly in the require or grow more rapidly in the

presence of increased pressurepresence of increased pressure

RadiationRadiation

Radiation damageRadiation damage

ionizing radiationionizing radiation– x rays and gamma raysx rays and gamma rays– mutations mutations death death– disrupts chemical structure of many disrupts chemical structure of many

molecules, including DNAmolecules, including DNA damage may be repaired by DNA repair damage may be repaired by DNA repair

mechanismsmechanisms

Radiation damage…Radiation damage…

ultraviolet (UV) radiationultraviolet (UV) radiation– mutations mutations death death– causes formation of thymine dimers in causes formation of thymine dimers in

DNADNA– DNA damage can be repaired by two DNA damage can be repaired by two

mechanismsmechanisms photoreactivationphotoreactivation – dimers split in presence of – dimers split in presence of

lightlight dark reactivationdark reactivation – dimers excised and replaced – dimers excised and replaced

in absence of lightin absence of light

Radiation damage…Radiation damage…

visible lightvisible light– at high intensities generates at high intensities generates singlet singlet

oxygenoxygen ( (11OO22)) powerful oxidizing agentpowerful oxidizing agent

– carotenoid pigmentscarotenoid pigments protect many light-exposed protect many light-exposed

microorganisms from photooxidationmicroorganisms from photooxidation

Microbial Growth in Microbial Growth in Natural EnvironmentsNatural Environments

microbial environments are microbial environments are complex, constantly changing, complex, constantly changing, and may expose a and may expose a microorganism to overlapping microorganism to overlapping gradients of nutrients and gradients of nutrients and environmental factorsenvironmental factors

Growth Limitation by Growth Limitation by Environmental FactorsEnvironmental Factors

Leibig’s law of the minimumLeibig’s law of the minimum– total biomass of organism determined total biomass of organism determined

by nutrient present at lowest by nutrient present at lowest concentrationconcentration

Shelford’s law of toleranceShelford’s law of tolerance– above or below certain environmental above or below certain environmental

limits, a microorganism will not grow, limits, a microorganism will not grow, regardless of the nutrient supplyregardless of the nutrient supply

Responses to low Responses to low nutrient levelsnutrient levels oligotrophic environmentsoligotrophic environments morphological changes to morphological changes to

increase surface area and ability increase surface area and ability to absorb nutrientsto absorb nutrients

mechanisms to sequester certain mechanisms to sequester certain nutrientsnutrients

Counting Viable but Counting Viable but Nonculturable Nonculturable Vegetative ProcaryotesVegetative Procaryotes

stressed microorganisms can stressed microorganisms can temporarily lose ability to grow using temporarily lose ability to grow using normal cultivation methodsnormal cultivation methods

microscopic and isotopic methods for microscopic and isotopic methods for counting viable but nonculturable cells counting viable but nonculturable cells have been developedhave been developed

Quorum Sensing and Quorum Sensing and Microbial PopulationsMicrobial Populations

quorum sensingquorum sensing– microbial microbial

communication communication and cooperationand cooperation

– involves involves secretion and secretion and detection of detection of chemical signalschemical signals

Figure 6.20

Processes sensitive to Processes sensitive to quorum sensing: gram-quorum sensing: gram-negative bacterianegative bacteria

bioluminescence (bioluminescence (Vibrio fischeriVibrio fischeri)) synthesis and release of virulence factors synthesis and release of virulence factors

((Pseudomonas aeruginosaPseudomonas aeruginosa)) conjugation (conjugation (Agrobacterium tumefaciensAgrobacterium tumefaciens)) antibiotic production (antibiotic production (Erwinia carotovora, Erwinia carotovora,

Pseudomonas aureofaciensPseudomonas aureofaciens)) biofilm production (biofilm production (P. aeruginosaP. aeruginosa))

Quorum sensing: gram-Quorum sensing: gram-positive bacteriapositive bacteria

often mediated by oligopeptide often mediated by oligopeptide pheromonepheromone

processes impacted by quorum sensing:processes impacted by quorum sensing:– mating (mating (Enterococcus faecalisEnterococcus faecalis))– transformation competence (transformation competence (Streptococcus Streptococcus

pneumoniaepneumoniae))– sporulation (sporulation (Bacillus subtilisBacillus subtilis))– production of virulence factors (production of virulence factors (Staphylococcus Staphylococcus

aureusaureus))– development of aerial mycelia (development of aerial mycelia (Streptomyces Streptomyces

griseusgriseus))– antibiotic production (antibiotic production (S. griseusS. griseus))

The Lux Gene in The Lux Gene in Vibrio Vibrio FischeriFischeri

Requirements for NitrogenRequirements for Nitrogen

Nitrogen is required for the synthesis of amino acids that Nitrogen is required for the synthesis of amino acids that compose the structure of proteins, purines and pyrimidines the compose the structure of proteins, purines and pyrimidines the bases of both DNA and RNA, and for other derivative molecules bases of both DNA and RNA, and for other derivative molecules such as glucosamine.such as glucosamine.

Many microorganisms can use the nitrogen directly from amino Many microorganisms can use the nitrogen directly from amino acids. The amino group ( NH2) is derived from ammonia acids. The amino group ( NH2) is derived from ammonia through the action of enzymes such as glutamate through the action of enzymes such as glutamate dehydrogenase.dehydrogenase.

Most photoautotrophs and many nonphotosynthetic Most photoautotrophs and many nonphotosynthetic microorganisms reduce nitrate to ammonia and assimilate microorganisms reduce nitrate to ammonia and assimilate nitrogen through nitrate reduction. A variety of bacteria are nitrogen through nitrate reduction. A variety of bacteria are involved in the nitrogen cycle such as involved in the nitrogen cycle such as RhizobiumRhizobium which is able which is able to use atmospheric nitrogen and convert it to ammonia. ( Found to use atmospheric nitrogen and convert it to ammonia. ( Found on the roots of legumes like soy beans and clover) These on the roots of legumes like soy beans and clover) These compounds are vital for the Nitrogen cycle and the incorporation compounds are vital for the Nitrogen cycle and the incorporation of nitrogen into plants to make nitrogen comounds.of nitrogen into plants to make nitrogen comounds.

PhosphorousPhosphorous

Phosphorous is present in phospholipids( Phosphorous is present in phospholipids( membranes), Nucleic acids( DNA and membranes), Nucleic acids( DNA and RNA), coenzymes, ATP, some proteins, RNA), coenzymes, ATP, some proteins, and other key cellular components. and other key cellular components.

Inorganic phosphorous is derived from Inorganic phosphorous is derived from the environment in the form of the environment in the form of phosphates. Some microbes such as phosphates. Some microbes such as E. E. colicoli can use organophosphates such as can use organophosphates such as hexose – 6-phosphates . hexose – 6-phosphates .

MixotrophyMixotrophy

Chemical energy – source organicChemical energy – source organic Inorganic H/e- donorInorganic H/e- donor Organic carbon sourceOrganic carbon source

Requirements for Requirements for Nitrogen, Phosphorus, Nitrogen, Phosphorus, and Sulfurand Sulfur

Needed for synthesis of important Needed for synthesis of important molecules (e.g., amino acids, nucleic molecules (e.g., amino acids, nucleic acids)acids)

Nitrogen supplied in numerous waysNitrogen supplied in numerous ways Phosphorus usually supplied as Phosphorus usually supplied as

inorganic phosphateinorganic phosphate Sulfur usually supplied as sulfate via Sulfur usually supplied as sulfate via

assimilatory sulfate reductionassimilatory sulfate reduction

Sources of nitrogenSources of nitrogen

organic moleculesorganic molecules ammoniaammonia nitrate via assimilatory nitrate nitrate via assimilatory nitrate

reductionreduction nitrogen gas via nitrogen fixationnitrogen gas via nitrogen fixation

Growth FactorsGrowth Factors

organic compoundsorganic compounds essential cell components (or essential cell components (or

their precursors) that the cell their precursors) that the cell cannot synthesizecannot synthesize

must be supplied by environment must be supplied by environment if cell is to survive and reproduceif cell is to survive and reproduce

Classes of growth Classes of growth factorsfactors amino acidsamino acids

– needed for protein synthesisneeded for protein synthesis purines and pyrimidinespurines and pyrimidines

– needed for nucleic acid synthesisneeded for nucleic acid synthesis vitaminsvitamins

– function as enzyme cofactorsfunction as enzyme cofactors

Amino acids

Proteins

Bases of nucleic acidsBases of nucleic acids Adenine and guanine Adenine and guanine

are purinesare purines

Cytosine, thymine, Cytosine, thymine, and uracil are and uracil are pyrimidinespyrimidines

Also found in energy Also found in energy triphosphates( ATP triphosphates( ATP and GTPand GTP))

Practical importance of Practical importance of growth factorsgrowth factors

development of quantitative development of quantitative growth-response assays for growth-response assays for measuring concentrations of measuring concentrations of growth factors in a preparationgrowth factors in a preparation

industrial production of growth industrial production of growth factors by microorganisms factors by microorganisms

Uptake of Nutrients Uptake of Nutrients by the Cellby the Cell Some nutrients enter by Some nutrients enter by passive passive

diffusiondiffusion Most nutrients enter by:Most nutrients enter by:

– facilitated diffusionfacilitated diffusion– active transportactive transport– group translocationgroup translocation

Passive DiffusionPassive Diffusion

molecules move from region of molecules move from region of higher concentration to one of higher concentration to one of lower concentration because of lower concentration because of random thermal agitationrandom thermal agitation

HH22O, OO, O22 and CO and CO22 often move often move across membranes this wayacross membranes this way

Active TransportActive Transport

energy-dependent processenergy-dependent process– ATP or proton motive force usedATP or proton motive force used

moves molecules against the moves molecules against the gradientgradient

concentrates molecules inside cellconcentrates molecules inside cell involves carrier proteins involves carrier proteins

(permeases)(permeases)– carrier saturation effect is observedcarrier saturation effect is observed

TransportersTransporters

“Molecular Properties of Bacterial Multidrug

Transporters” – Monique Putnam, Hendrik van Veen, and Wil Konings – PubMed Central. Full Text available .

Microbiol Mol Biol Review. 2000 December; 64 (4): 672–693

ABC transportersABC transporters

ATP-binding ATP-binding cassette cassette transporterstransporters

observed in observed in bacteria, bacteria, archaea, archaea, and and eucaryoteseucaryotes

Figure 5.3

Figure 5.4

antiport

symport

Group TranslocationGroup Translocation molecules are molecules are

modified as modified as they are they are transported transported across the across the membranemembrane

energy-energy-dependent dependent processprocess

Figure 5.5

Fe uptake in Fe uptake in pathogenspathogens The ability of pathogens to obtain iron The ability of pathogens to obtain iron

from transferrins, ferritin, hemoglobin, and from transferrins, ferritin, hemoglobin, and other iron-containing proteins of their host other iron-containing proteins of their host is central to whether they live or dieis central to whether they live or die

Some invading bacteria respond by Some invading bacteria respond by producing specific iron chelators - producing specific iron chelators - siderophores that remove the iron from the siderophores that remove the iron from the host sources. Other bacteria rely on direct host sources. Other bacteria rely on direct contact with host iron proteins, either contact with host iron proteins, either abstracting the iron at their surface or, as abstracting the iron at their surface or, as with heme, taking it up into the cytoplasm with heme, taking it up into the cytoplasm

Iron and signallingIron and signalling

Iron is also used by pathogenic bacteria Iron is also used by pathogenic bacteria as a signal molecule for the regulation of as a signal molecule for the regulation of virulence gene expression. This sensory virulence gene expression. This sensory system is based on the marked system is based on the marked differences in free iron concentrations differences in free iron concentrations between the environment and intestinal between the environment and intestinal lumen (high) and host tissues (low)lumen (high) and host tissues (low)

ListeriaListeria Pathogenesis and Molecular Virulence Determinants Pathogenesis and Molecular Virulence Determinants

José A. Vázquez-Boland,1,2* Michael Kuhn,3 Patrick Berche,4 Trinad Chakraborty,5 José A. Vázquez-Boland,1,2* Michael Kuhn,3 Patrick Berche,4 Trinad Chakraborty,5 Gustavo Domínguez-Bernal,1 Werner Goebel,3 Bruno González-Zorn,1 Jürgen Gustavo Domínguez-Bernal,1 Werner Goebel,3 Bruno González-Zorn,1 Jürgen Wehland,6 and Jürgen Kreft3Wehland,6 and Jürgen Kreft3

Pathogens and Iron Pathogens and Iron uptakeuptake

Burkholderia cepaciaBurkholderia cepacia Campylobacter jejuniCampylobacter jejuni Pseudomonas aeruginosaPseudomonas aeruginosa E. coliE. coli Listeria monocytogenesListeria monocytogenes

Iron UptakeIron Uptake ferric iron is very ferric iron is very

insoluble so uptake insoluble so uptake is difficultis difficult

microorganisms use microorganisms use siderophoressiderophores to aid to aid uptakeuptake

siderophore siderophore complexes with ferric complexes with ferric ionion

complex is then complex is then transported into celltransported into cell

Figure 5.6

ListeriosisListeriosis

One involves the direct transport One involves the direct transport of ferric citrate to the bacterial cell of ferric citrate to the bacterial cell

Another system involves an Another system involves an extracellular ferric iron reductase, extracellular ferric iron reductase, which uses siderophores which uses siderophores

The third system may involve a The third system may involve a bacterial cell surface-located bacterial cell surface-located transferrin-binding proteintransferrin-binding protein

Iron bacteria in the Iron bacteria in the environmentenvironment There are several non-disease producing There are several non-disease producing

bacteria which grow and multiply in water bacteria which grow and multiply in water and use dissolved iron as part of their and use dissolved iron as part of their metabolism. They oxidize iron into its metabolism. They oxidize iron into its insoluble ferric state and deposit it in the insoluble ferric state and deposit it in the slimy gelatinous material which surrounds slimy gelatinous material which surrounds their cells. their cells.

These filamentous bacteria grow in stringy These filamentous bacteria grow in stringy clumps and are found in most iron-bearing clumps and are found in most iron-bearing surface waters. They have been known to surface waters. They have been known to proliferate in waters containing iron as low proliferate in waters containing iron as low as 0.1 mg/l. as 0.1 mg/l.

Culture MediaCulture Media preparations devised to support preparations devised to support

the growth (reproduction) of the growth (reproduction) of microorganismsmicroorganisms

can be liquid or solidcan be liquid or solid– solid media are usually solidified with solid media are usually solidified with

agaragar important to study of important to study of

microorganismsmicroorganisms

Synthetic or Defined Synthetic or Defined MediaMedia

all all components components and their and their concentrations concentrations are knownare known

Complex MediaComplex Media contain some contain some

ingredients of ingredients of unknown unknown composition composition and/or and/or concentrationconcentration

Some media Some media componentscomponents peptonespeptones

– protein hydrolysates prepared by partial protein hydrolysates prepared by partial digestion of various protein sourcesdigestion of various protein sources

extractsextracts– aqueous extracts, usually of beef or yeastaqueous extracts, usually of beef or yeast

agaragar– sulfated polysaccharide used to solidify sulfated polysaccharide used to solidify

liquid medialiquid media

Types of MediaTypes of Media

general purpose mediageneral purpose media– support the growth of many support the growth of many

microorganismsmicroorganisms– e.g., tryptic soy agare.g., tryptic soy agar

enriched mediaenriched media– general purpose media supplemented by general purpose media supplemented by

blood or other special nutrientsblood or other special nutrients– e.g., blood agare.g., blood agar

Types of media…Types of media…

Selective mediaSelective media– Favor the growth of some Favor the growth of some

microorganisms and inhibit growth microorganisms and inhibit growth of othersof others

– MacConkey agarMacConkey agar selects for gram-negative bacteriaselects for gram-negative bacteria Inhibits the growth of gram-positive Inhibits the growth of gram-positive

bacteriabacteria

Beta Hemolysis

Types of media…Types of media… Differential mediaDifferential media

– Distinguish between different groups Distinguish between different groups of microorganisms based on their of microorganisms based on their biological characteristicsbiological characteristics

– Blood agarBlood agar hemolytic versus nonhemolytic bacteriahemolytic versus nonhemolytic bacteria

– MacConkey agarMacConkey agar lactose fermenters versus nonfermenterslactose fermenters versus nonfermenters

Selective and differential media

Selects for Gram –

Differentiates between bacteria based upon fermentation of lactose( color change)

Organism

Salt Tolerance

Mannitol Fermentation

 1. S. aureus

Positive - growth

Positive (yellow)

 2. S. epidermidis

Positive*- growth

Negative( color does not change) – no fermentation of mannitol with production of acid

 3. M. luteus

Negative

N/A**

http://www.austin.cc.tx.us/microbugz/20msa.html

 

Web References on Media

 

http://www.jlindquist.net/generalmicro/102diff.html - General Reference

http://medic.med.uth.tmc.edu/path/macconk.htm - MacConkey Agar

http://www.indstate.edu/thcme/micro/hemolys.html - Blood Agar

 

Figure 5.7

1. dispense cells ontomedium in petri dish

2. - 3. sterilize spreader

4. spread cellsacross surface

Spread-plate Spread-plate techniquetechnique

Figure 5.8

inoculatingloop

Streak plate techniqueStreak plate technique

Isolation of Pure Isolation of Pure CulturesCultures Pure culturePure culture

– population of cells arising from a population of cells arising from a single cellsingle cell

Spread plateSpread plate, , streak platestreak plate, and , and pour platepour plate are techniques used to are techniques used to isolate pure culturesisolate pure cultures

Colony Morphology and Colony Morphology and GrowthGrowth

individual individual species form species form characteristic characteristic coloniescolonies

Figure 5.10b

Terms1. Colony shape and size: round, irregular, punctiform (tiny)2. Margin (edge): entire (smooth), undulate (wavy), lobate (lobed)3. Elevation: convex, umbonate, flat, raised4. Color: color or pigment, plus opaque, translucent, shiny or dull5. Texture: moist, mucoid, dry (or rough).

Figure 5.10a

Colony growthColony growth

Most rapid at edge of colonyMost rapid at edge of colony– oxygen and nutrients are more oxygen and nutrients are more

available at edgeavailable at edge Slowest at center of colonySlowest at center of colony In nature, many microorganisms In nature, many microorganisms

form biofilms on surfacesform biofilms on surfaces